PROJECT SUMMARY My proposed work aims at elucidating fundamental molecular mechanisms underlying Homeobox-only protein (HOPX) function in cardiac lineage commitment and the role of nuclear organization in shaping cell fate decisions. Progressive lineage restriction occurs as undifferentiated cells develop into mature cell types. During cardiogenesis, multipotent cardiac progenitors (CPCs), marked by Isl1 and Nkx2-5 expression, give rise to endothelial, smooth muscle, and cardiomyocyte lineages. Work by our laboratory demonstrated that Hopx expression is downstream of Nkx2-5 and defines a pool of CPCs that exclusively gives rise to cardiomyocytes. However, it unknown if and how Hopx functions to restrict cell fate choice during cardiomyocyte lineage commitment. HOPX is an atypical homeodomain-containing protein that lacks DNA binding capacity. Interestingly, recent studies suggest that it may function as a transcriptional co-repressor, in part by recruiting histone deacetylases to affect gene expression. Consistent with its repressive role, Hopx deletion during cardiac differentiation of embryonic stem cells results in aberrant expression of genes relevant to unwanted lineages. Recent data from our laboratory also demonstrates that key developmentally-regulated cardiac genes are released from the nuclear periphery upon differentiation cardiac myocytes, adding to the mounting evidence that the spatial organization of chromatin guides cell differentiation. Excitingly, preliminary experiments indicate that Hopx interacts with nuclear lamina proteins. In this context, I hypothesize that HOPX regulates cardiac myocyte commitment and coordinates spatial positioning of the genome to restrict alternative lineage choices. Using a genetic lineage tracing approach, I aim to define the role of Hopx during cardiomyocyte commitment. Studies to date suggest that a subset Hopx-/- progenitors cannot faithfully commit to the myocyte lineage and instead adopt an endothelial cell fate in vivo. In addition, I aim to define regions of the genome that associate with the nuclear lamina in murine cardiac myocytes and define changes upon loss of Hopx during cardiogenesis. Taken together, our results will advance our understanding of how HOPX role in myocyte commitment and nuclear lamina-chromatin interactions, thereby providing a window into how spatial organization of the genome impacts coordinated gene regulation and cell fate choices.